Mossbauer and Electron Paramagnetic Resonance Studies of Chloroperoxidase Following Mechanism-Based Inactivation with Allylbenzene

We have used Mossbauer and electron paramagnetic resonance (EPR) spectroscopy to study a heme-N-alkylated derivative of chloroperoxidase (CPO) prepared by mechanism-based inactivation with allylbenzene and hydrogen peroxide. The freshly prepared inactivated enzyme (``green CPO'') displayed a nearly pure low-spin ferric EPR signal with g = 1.94,2.15,2.31. The Mossbauer spectrum of the same species recorded at 4.2 K showed magnetic hyperfine splittings, which could be simulated in terms of a spin Hamiltonian with a complete set of hyperfine parameters in the slow spin fluctuation limit. The EPR spectrum of green CPO was simulated using a three-term crystal field model including g-strain. The best-fit parameters implied a very strong octahedral field in which the three2T2levels of the (3d)5configuration in green CPO were lowest in energy, followed by a quartet. In native CPO, the6A1states follow the2T2ground state doublet. The alkene-mediated inactivation of CPO is spontaneously reversible. Warming of a sample of green CPO to 22 degrees C for increasing times before freezing revealed slow conversion of the novel EPR species to two further spin S = 1/2 ferric species. One of these species displayed g = 1.82, 2.25, 2.60 indistinguishable from native CPO. By subtracting spectral components due to native and green CPO, a third species with g = 1.86, 2.24, 2.50 could be generated. The EPR spectrum of this ``quasi-native CPO,'' which appears at intermediate times during the reactivation, was simulated using best-fit parameters similar to those used for native CPO.